Coronavirus pseudovirus packaging system, packaging method therefor, and application of coronavirus pseudovirus in evaluating disinfection efficacy

The present application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on Jun. 16, 2023, is named Substitute Sequence Listing_ST25.txt and is 71,623 bytes in size.

This disclosure relates to the field of gene and cell engineering and virology, and in particular, to a pseudovirus packaging vector and a packaging cell system. The pseudovirus packaging vector and the packaging system may be used for preparing a coronavirus pseudovirus by a one-step packaging method, and the prepared coronavirus pseudovirus may be used as a biological indicator for detection and evaluation of efficacy of a biological and chemical substance and a physical treatment method for inhibiting and disinfecting coronavirus.

COVID-19 is a coronavirus that can cause fatal pneumonia in humans. In addition to COVID-19, two other coronaviruses, severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), also have caused fatal pneumonia in humans since the early 21st century. In addition, four low-pathogenic coronaviruses are also prevalent in humans: HCoV-OC43, HCoV-HKU1, HCoV-NL63, and HCoV-229E. Local cases of COVID-19 infection have been confirmed in many places in China. It has been confirmed that transmission of COVID-19 can occur frequently through cold chain logistics processes. In accordance with the COVID-19 Prevention and Control Plan issued by the State Council of RPC, all local governments are required to take protection actions for people engaged in cold-chain food operations. Cutting off transmission of COVID-19 through cold chain logistics processes directly affects China's overall coordinated approach to the decisions of epidemic control as well as strengthening of the strategy of “preventing the coronavirus from re-entering the country to cause a new epidemic”.

Virus inactivation, a conventional technology, may be simply divided into physical inactivation and chemical inactivation, based on the principle thereof. Although there is a variety of common virucidal disinfectants such as ozone, ultraviolet light, chlorine dioxide, peracetic acid, hydrogen peroxide, sodium dichloroisocyanurate, irradiation, negative ions, such virucidal disinfectants have inconsistent capability of virus inactivation, and application ranges of such virucidal disinfectants have no data support, mainly because there is no unified and effective method for evaluating capability of a virucidal disinfectant for virus inactivation. Particularly, for SARS-CoV-2, SARS-CoV, MERS-CoV and other highly transmissible and harmful viruses, evaluation of live viruses may only be carried out in a P3 laboratory. The lack of test and evaluation methods with biological safety directly leads to the lack of evaluation support for suitable new technologies or applications such as ozone and irradiation, which further makes it hard to determine parameters and applicable rules of the technologies, as well as promotion and application of the technologies.

The known pseudovirus refers to a replication-defective virus that is capable of integrating an envelope glycoprotein of another different virus to form a virus with the envelope of the exogenous virus, while its genome keeps the genomic characteristics of the original virus vector itself. Pseudoviruses have lost their replication capability due to genetic defect in their genome, only capable of single-cycle infection, thus have high biological safety. Therefore, the pseudovirus approach can provide a safe and effective research method for studying viruses that are highly pathogenic, highly infectious, or hard to be cultured in vitro, such as SARS-CoV-2. Furthermore, pseudoviruses constructed in vitro also have advantages such as high stability and wide host tropism, so they are widely used in research, development, and evaluation of vaccines; detection, screening, and evaluation of neutralizing antibodies; discovery of antigenic epitopes of neutralizing antibodies; research and development of antibodies, macromolecule and small-molecule drugs to inhibit virus invasion, as well as physically virucidal disinfection methods and chemical virucidal disinfectants.

It is of great significance to package and prepare pseudoviruses having no potential biosafety hazards for SARS-CoV-2, SARS-CoV, MERS-CoV, and the like, and to apply them to examine and evaluate virus neutralization and inhibition capability, as well as the performance and efficacy of virucidal disinfectants in virus inactivation.

It is safe and effective to use pseudoviruses that are replication-defective and unable to produce infectious progeny to simulate a process of a wild virus infecting a host. Packaging of VSV occurs on the cell membrane and involves virion budding from the cell surface. During the budding, VSV acquires an envelope formed by a bilayer lipid from the cell membrane and a trimer of VSV glycoprotein (VSV-G). When VSV-G genes are partially or completely replaced by dual-reporter genes, and an envelope protein of an exogenous virus is fully expressed in dVSVΔG-infected cells, the exogenous virus glycoprotein can be assembled onto the envelope of VSV virus particles. In this study, we compared pseudoviruses packaged with the full-length COVID-19 S protein (COVID-19-S), C-terminal truncated S protein (C19-HA), and S protein with 19aa truncated at C-terminal and fused with HA (C19-HA) dVSVΔG-COVID-19-S-C19-HA. It is found that pseudovirus dVSVΔG-COVID-19-S-C19-HA has a much higher infection efficiency than that of dVSVΔG-COVID-19-S, and also significantly higher than that of dVSVΔG-COVID-19-S-C19. In addition, 293T-hACE2 cells stably expressing human ACE2 (hACE2) are most sensitive to pseudovirus infection. In contrast, Vero-E6 has moderate infection efficiency, while 293 T cells are completely insensitive to COVID-19 pseudoviruses. Based on dVSVΔG-COVID-19-S-C19-HA and 293T-hACE2 cells, methods for detection and evaluation of efficacy of biological, chemicals and physical treatment methods for inhibiting and disinfecting coronavirus (including evaluation of efficacy of ozone in disinfecting coronavirus in cold chain environment), and evaluation of neutralization activity of antibodies in serum samples after immunization with various coronavirus vaccines are established. This method system can be used for the research of coronavirus such as COVID-19 (SARS-CoV-2), SARS (SARS-CoV), MERS, and the like.

A coronavirus pseudovirus packaging system comprises a safe and efficient modified vesicular stomatitis virus (VSV), and an packaging cell that expresses coronavirus spike protein; wherein the modified vesicular stomatitis virus VSV is defined as a replication-defective virus with GP gene partially or completely replaced by Fluc and EGFP dual-reporter genes, and the modified vesicular stomatitis virus VSV is named as dVSVΔG-Fluc-EGFP.

Preferably, in the coronavirus pseudovirus packaging system, the dual-reporter genes include a fluorescent protein reporter gene and a luciferase reporter gene, and the fluorescent protein reporter gene is selected from a reporter gene corresponding to green fluorescent protein or red fluorescent protein; and the luciferase reporter gene is selected from a reporter gene corresponding to firefly luciferase or renilla luciferase.

Preferably, in the coronavirus pseudovirus packaging system, the fluorescent protein reporter gene is an enhanced green fluorescent protein (EGFP) gene, and the corresponding gene sequence is set forth in SEQ ID NO: 1; the luciferase reporter gene is selected from firefly luciferase Fluc gene with optimized codons, and the sequence of the firefly luciferase Fluc gene is set forth in SEQ ID NO: 2.

Preferably, in the coronavirus pseudovirus packaging system, the gene encoding GP in the genetic material of dVSVΔG-Fluc-EGFP is replaced by the Fluc reporter gene, the EGFP reporter gene is integrated between Fluc and VSV polymerase L gene, and the gene sequence of dVSVΔG-Fluc-EGFP is set forth in SEQ ID NO: 3.

Preferably, in the coronavirus pseudovirus packaging system, the packaging cell is selected from 293T, the packaging cell transiently or stably or inductively expresses the coronavirus spike protein, the transient expression is realized by transfecting the cell with an eukaryotic expression vector, the stable expression is realized by transducing the cell with a lentiviral vector system, and the corresponding induced expression is realized by transducing the cell with a tetracycline-regulated tet-on/off vector system.

Preferably, in the coronavirus pseudovirus packaging system, the envelope protein expressed by the packaging cell corresponds to the S gene in the coronaviruses SARS, MERS or COVID-19, and the SARS coronavirus envelope protein is selected from a sequence obtained after deletion of 19 amino acids at 3′ end of the SARS coronavirus spike protein, namely a sequence of SEQ ID NO: 4; the MERS coronavirus envelope protein is selected from a sequence obtained after deletion of 19 amino acids at 3′ end of the MERS coronavirus spike protein, namely a sequence of SEQ ID NO: 5; the COVID-19 coronavirus envelope protein is selected from a sequence obtained after deletion of 19 amino acids at 3′ end of the COVID-19 coronavirus spike protein, namely a sequence of SEQ ID NO: 6, or the COVID-19 coronavirus envelope protein is selected from a sequence obtained after deletion of 19 amino acids at 3′ end of the COVID-19 coronavirus spike protein and fusion of HA protein, namely a sequence of SEQ ID NO: 7; the envelope protein expression expressed by the packaging cell is mediated by transient expression plasmid or stable expression plasmid or stable and inducible expression lentivirus vector, including eukaryotic expression vector, and four plasmids transiently expressing the envelope protein in the packaging cell are named as expression plasmids pCA-SARS-C19, pCA-MERS-C19, pCA-COVID-19-C19, and pCA-COVID-19-C19-HA, respectively, as well as derivative vector expressing the same encoded protein. Preferably, the target to be fused after deletion of 19aa at the 3′ end of S in the S-C19-HA is not limited to HA, but may be other peptides with labeling function such as flag, myc, and his.

A one-step packaging method for a pseudovirus packaging system, wherein the pseudovirus packaging system includes dVSVΔG-Fluc-EGFP and an packaging cell that expresses the coronavirus spike protein, wherein the surface of dVSVΔG-Fluc-EGFP is assembled with complete GP envelope protein, GP gene in genetic material is partially or completely replaced by Fluc and EGFP dual-reporter genes, the expression of the coronavirus spike protein is mediated by pCAGGS, the coronavirus spike protein is selected from a truncate of 16aa-28aa at 3′ end of the S gene, and dVSVΔG-Fluc-EGFP and the packaging cell that expresses the coronavirus spike protein are mixed in one step, and supernatant is collected after a certain time to obtain the coronavirus pseudovirus. Preferably, the coronavirus spike protein performs best when it is selected from a truncate of 19aa at 3′ end of the S gene.

Preferably, the one-step packaging method includes the following steps:

An additional pseudovirus packaging system comprises modified vesicular stomatitis virus VSV, an packaging cell that expresses spike protein of an additional virus; wherein the modified vesicular stomatitis virus VSV is defined as a VSV replication-defective virus with GP gene partially or completely replaced by Fluc and EGFP dual-reporter genes, and the VSV replication-defective virus is named as dVSVΔG-Fluc-EGFP. The transiently expressed or stably expressed or inductively expressed packaging plasmid and vector express an envelope protein of a target virus in an packaging cell, and the envelope protein expressed at the cell level by the transiently expressed or stably expressed or inductively expressed packaging plasmid and vector is selected from one or more of coronavirus, herpesvirus, rhabdovirus, poxvirus, hepadnavirus, filovirus, rhabdovirus, influenza virus, paramyxovirus, flavivirus, paramyxovirus, flavivirus, enveloped virus, bunyavirus, or retrovirus.

Preferably, in the additional pseudovirus packaging system, the coronavirus is COVID-19 (SARS-CoV-2), SARS (SARS-CoV), MERS (MERS-CoV), HCoV-OC43, HCoV-HKU1, HCOV-NL63, or HCoV-229E; the hepadnavirus is hepatitis B virus or hepatitis C virus; the filovirus is Ebola virus; the rhabdovirus is rabies virus; the paramyxovirus is measles virus or respiratory syncytial virus; and the flavivirus is Zika virus or dengue virus.

Preferably, the additional pseudovirus packaging system includes dVSVΔG-Fluc-EGFP and the packaging cell, and the packaging cell is selected from 293, 293T, 293sus, HEK293, HEK293T, HEK293FT, BHK, or Vero with high transfection efficiency and good stability. That is, the pseudovirus packaging system of this disclosure may be applied to a variety of viruses other than those described in detail herein, and may be widely applied to preparation of pseudoviruses for other virus types.

The coronavirus pseudoviruses packaged by the above coronavirus pseudovirus packaging system may be used as a biological indicator to replace a wild-type coronavirus for detection and evaluation of efficacy of biological and chemical substances and physical treatment methods for inhibiting and disinfecting the coronavirus, wherein the substances and the methods for inhibiting and disinfecting the coronavirus include an anti-coronavirus neutralizing antibody and medicament, chemical virucidal disinfectants and physical virucidal disinfection means.

Use of a coronavirus pseudovirus in evaluation of efficacy of a virucidal disinfectant, comprises the following steps:

Preferably, the use of the coronavirus pseudovirus in evaluation of the efficacy of the virucidal disinfectant is characterized in that the virus-contaminated environment in the step (1) includes a logistics environment (such as a cold-chain logistics environment), a home environment, a public place, a school environment and the like.

Preferably, the use of the coronavirus pseudovirus in evaluation of the efficacy of the virucidal disinfectant is characterized in that multiple experimental groups may be constructed in the step (1) to avoid excessive errors, and the step (3) includes observing the expression of fluorescent protein and luciferase after 293T-hACE2 is infected by the pseudovirus for measurement and calculation of infection capacity and bioactivity titer (PFU/ml) of the pseudovirus as well as detection of copy number of the pseudovirus nucleic acid.

Preferably, the use of the coronavirus pseudovirus in evaluation of the efficacy of the virucidal disinfectant is characterized in that the virucidal disinfectant (peroxides, quaternary ammonium salts, chlorine-containing compounds, and alcohols) and the physical treatment method in the step (3) includes various combinations of one or more of ozone, peroxyacetic acid, hydrogen peroxide, chlorine dioxide, oxydol, sodium dichloroisocyanurate, ultraviolet light, negative ions, irradiation, or the like.

Preferably, the use of the coronavirus pseudovirus in evaluation of the efficacy of the virucidal disinfectant is characterized in that the medium in the step (1) or step (3) includes one or more of plastic, foam, bookbinding paperboard, boxboard, textile, or metal foil.

By using the packaging system and the packaging method of this disclosure, pseudoviruses of various viruses with envelope proteins can be packaged, and these pseudoviruses, in a one-to-one correspondence with the viruses, can be safely, quickly and accurately used for detection and evaluation of efficacy of biological and chemical substances and physical treatment methods used for inhibiting and disinfecting coronavirus. The coronavirus pseudovirus of this disclosure can rapidly package a pseudovirus with single-cycle infection, low background signal and high titer, and has characteristics of rapid detection and simple and convenient operation compared with a lentivirus-based pseudovirus system. The pseudovirus packaging system of this disclosure has universal extendibility, is not limited to the coronavirus specifically described herein, but also can be extended to other types of viruses, and the one-step packaging method of the corresponding pseudovirus packaging system can also be applied to one-step packaging methods for other types of pseudoviruses.

The pseudovirus (not limited to coronavirus pseudovirus) packaged by the above packaging system and the packaging method may be used for detection and evaluation of efficacy of biological and chemical substances and physical treatment methods for inhibiting and disinfecting corresponding viruses. This disclosure has the following beneficial effects: (1) through optimal combination and design on the use environment and dosage of the detected substances and methods for inhibiting and disinfecting the coronavirus, the virus inactivation function of the detected substances and methods for inhibiting and disinfecting the coronavirus can be accurately, visually, qualitatively or quantitatively compared and verified; (2) the pseudovirus reporting system biological indicator in this disclosure has high biological safety, can simulate the transmission and pathogenic characteristics of various viruses, and can meet the requirements for evaluating the virus inactivation ability of different substances and methods for inhibiting and disinfecting viruses in conventional biosafety environment; (3) the pseudovirus fluorescent reporter gene in this disclosure can intuitively reflect the virus inactivation results of the detected substances and methods for inhibiting and disinfecting viruses; and (4) the use of this disclosure greatly promotes research and development of substances and methods for inhibiting and disinfecting viruses and studying the blocking effect on cold chain transmission, and provides practical and feasible favorable conditions.

COVID19 in the figures represents COVID-19.

In the following, this disclosure will be further described in detail with reference to specific examples, which are intended for explanation but not limitation of this disclosure. This disclosure mainly relates to integrating the genes of COVID-19-S, SARS-CoV-S, and MERS-S or truncated sequences thereof into an expression system, and further integrating VSV pseudovirus of the envelope antigen through the constructed dVSVΔG-EGFP-FLuc dual-reporter packaging system, and is used for detecting production of neutralizing antibodies in immune serum obtained after the relevant antigen protein is immunized in mice.

The reagents and consumables used in this disclosure are as follows: Lipofectamine LTX (Invitrogen 15338100), PBS (Hyclone SH30256.01), DMEM high glucose medium (Gibco C11995500), Penicillin-Streptomycin (Gibco 15140-122), fetal bovine serum (Gibco 10091-148), Opti-MEM® I Reduced Serum Medium (Gibco 31985-070), 96-well cell culture plate (Corning 3599), 6-well cell culture plate (Corning 3516), 6-cm cell culture plate (Corning 430166), COVID-19 RBD protein (Genescript Biotechnology Ltd Z03485), COVID-19 S1 protein (Genescript Biotechnology Ltd Z03485), SARS-CoV S RBD protein (Sino Biological 40150-V08B2), and MERS-CoV S1 protein (Sino Biological 40069-V08B1).

Cell Line:

Vero-E6 (ATCC, CRL-1586), 293T (ATCC-derived) cells were maintained in a high glucose DMEM (SIGMA-ALDRICH) and supplemented with 10% LBS (Gibco), penicillin (100 IU/mL), and streptomycin (100 μg/mL), passaged every 2 days in 5% carbon dioxide atmosphere at 37° C., infected with lentivirus expressing hACE2 for 72 hours, and screened for purinomycin resistance to obtain 293T-hACE2 cells.

Antibody Preparation:

Balb/C mice were immunized with COVID-19 spike protein (RBD/S1), SARS-CoV S RBD, and MERS-CoV S1 at 50 μg/mouse every other week. Complete adjuvant was added to the primary immunization, and incomplete adjuvant was added to the subsequent booster immunization to prepare specific polyclonal antibody against spike proteins of COVID-19, SARS-CoV and MERS-CoV, and activity of neutralizing antibody was identified.

Construction of Different Modified Envelope Expression Vectors:

Molecular construction: After codon optimization focusing on Spike protein (S protein) clone of COVID-19, full-length sequence of S (1-3822 bp), a sequence of S with 19 amino acids deleted from C-terminal (1-3765 bp), a sequence of S with 19 amino acids deleted from C-terminal plus HA tag (1-3792 bp), a sequence of S with 27 amino acids deleted from C-terminal (1-3735 bp), and a sequence of S with 53 amino acids deleted from C-terminal (1-3663 bp) were cloned into pCAGGS vector, respectively. For SARS-CoV and MERS-CoV, sequences of S with 19 amino acids deleted from C-terminal were selected as the first choice.

The S gene sequence published according to NCBI was codon optimized to facilitate the expression in cells. The sequence was respectively synthesized on a pCDNA3.1 vector by GenScript Biotech Corporation. After the target gene was amplified by PCR, the target band was recovered and purified by a fragment purification kit. The fragment and pCAGGS vector were digested with restriction endonucleases MCS1 (Xhol) and MCS2 (Nhel) at 37° C. for 3 h. The vector and the target fragment were recovered from gel, subjected to ligation reaction, and then transformed into competent cells. The positive clones were screened by colony PCR, the plasmid construction was verified by enzyme digestion and sequencing. The specific steps were as follows: 1. Primer synthesis and primer information: the primers were synthesized by GENEWIZ, Inc., and the PCR primers selected for construction and amplification of COVID-19-S gene are shown in Table 1.

1.1 The selected PCR primers and Colony PCR primers for amplification of COVID-19-S-C19 gene are shown in Table. 2:

1) The selected PCR primers and Colony PCR primers for amplifying COVID-19-S-C27 gene are shown in Table 3.

2) The selected PCR primers and Colony PCR primers for amplifying COVID-19-S-C53 gene are shown in Table 4.

3) The selected PCR primers and Colony PCR primers for amplifying COVID-19-S-C19HA gene are shown in Table 5.

4) The selected PCR primers and Colony PCR primers for amplifying SARS-COV-S-C19 gene are shown in Table 6.

5) The selected PCR primers and Colony PCR primers for amplifying MERS-CoV-S-C19 gene are shown in Table 7.

6) Target gene acquisition: PCR amplification was performed by using pCDNA3.1 plasmid with the target gene sequence as a template and using primers in Table 4.